guanosine-triphosphate and formic-acid

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.

Topics: Amino Acid Sequence; Animals; Binding Sites; Catalysis; Crystallization; Crystallography, X-Ray; Escherichia coli; Formamides; Formates; GTP Cyclohydrolase; Guanosine Triphosphate; Hydrogen Bonding; Hydrolysis; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Neopterin; Protein Conformation; Pteridines; Stereoisomerism; Zinc

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.

Topics: Catalysis; Escherichia coli; Formates; GTP Cyclohydrolase; Guanosine Triphosphate; Hydrolysis; Kinetics; Mutation; Neopterin; Pteridines; Pyrimidine Nucleotides; Schiff Bases; Spectrophotometry, Ultraviolet; Stereoisomerism

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.

Topics: Aldose-Ketose Isomerases; Escherichia coli; Flow Injection Analysis; Formates; GTP Cyclohydrolase; Guanosine Triphosphate; Models, Chemical; Neopterin; Pteridines; Pyrimidine Nucleotides

The experimental antineoplastic agent triciribine (tricyclic nucleoside, TCN) is known to be activated to its phosphate TCN-P by adenosine kinase and to inhibit cell growth, purine nucleotide synthesis, and incorporation of amino acids into proteins. Our objective in this paper was to compare these effects in intact cells of a human cell line as a prerequisite to describing in a companion paper [Moore et al., Biochem. Pharmac. 38, 4045 (1989)] more detailed enzymic studies of their interrelationships. TCN treatment inhibited cloning of CCRF-CEM human leukemic lymphoblasts 50% at concentrations of 6, 30, and 90 microM with 8-day, 8-hr, and 2-hr exposures respectively. However, 6-20% of the cells survived exposure to 200 microM TCN for 24 hr. The intracellular formation of TCN-P from TCN was rapid, concentrative and essentially complete, but TCN-P did not exceed about 1.4 mM (1.4 nmol/10(6) cells) at 200 microM TCN. In cells exposed to 50 microM TCN for 1.25 to 24 hr, formate incorporation into ATP and GTP was inhibited the most rapidly and strongly; pools of ATP and GTP were decreased as much as 40% (as compared with controls); and incorporation of formate into RNA purines was inhibited as much as 65%. Incorporation of leucine into protein was more moderately inhibited up to 40%, apparently in proportion to the concentration of intracellular TCN-P, rather than of the TCN in the medium. These inhibitions occurred most rapidly during the first 2-4 hr and increased only gradually thereafter, whereas cloning ability was inhibited more slowly and uniformly over a longer time period. No one of these metabolic effects by itself showed a clear correlation with the loss of viability. The incorporation of formate into formylglycinamide ribotide (FGAR, when accumulated at a blockage by azaserine) was inhibited drastically by TCN. The rate of incorporation of hypoxanthine into ATP was increased by TCN, whereas incorporation into GTP was decreased. Thus, the principal sites of inhibition of purine synthesis by TCN-P were shown in these intact cells to be at a step prior to synthesis of FGAR in the de novo pathway and also at an additional site between IMP and GTP.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Cell Survival; Formates; Glycine; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; Kinetics; Leukemia, Lymphoid; Lymphocytes; Phosphorylation; Protein Biosynthesis; Purines; Ribonucleosides; Ribonucleotides; RNA; Tumor Cells, Cultured

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.

Topics: Alkaline Phosphatase; Animals; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Female; Formates; Guanine; Guanosine; Guanosine Triphosphate; Hypoxanthine Phosphoribosyltransferase; Inosine; Ovary; Thymidine